Production of a high-efficiency TILLING population through polyploidization.
Helen Tsai 1, Victor Missirian, Kathie J Ngo, Robert K Tran, Simon R Chan, Venkatesan Sundaresan, Luca Comai
Targeting Induced Local Lesions in Genomes (TILLING) provides a nontransgenic method for reverse genetics that is widely applicable, even in species where other functional resources are missing or expensive to build. The efficiency of TILLING, however, is greatly facilitated by high mutation density. Species vary in the number of mutations induced by comparable mutagenic treatments, suggesting that genetic background may affect the response. Allopolyploid species have often yielded higher mutation density than diploids. To examine the effect of ploidy, we autotetraploidized the Arabidopsis (Arabidopsis thaliana) ecotype Columbia, whose diploid has been used for TILLING extensively, and mutagenized…
Plant Physiol. 2013 Apr;161(4):1604-14. doi: 10.1104/pp.112.213256. Epub 2013 Feb 15.PMID: 23417087
Discovery of rare mutations in populations: TILLING by sequencing.
Helen Tsai 1, Tyson Howell, Rebecca Nitcher, Victor Missirian, Brian Watson, Kathie J Ngo, Meric Lieberman, Joseph Fass, Cristobal Uauy, Robert K Tran, Asif Ali Khan, Vladimir Filkov, Thomas H Tai, Jorge Dubcovsky, Luca Comai
Discovery of rare mutations in populations requires methods, such as TILLING (for Targeting Induced Local Lesions in Genomes), for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum), we developed a method based on Illumina sequencing of target genes amplified from multidimensionally pooled templates representing 768 individuals per experiment. Parallel processing of sequencing libraries was aided by unique tracer sequences and barcodes allowing flexibility in the number and pooling arrangement of targeted genes, species, and pooling scheme. Sequencing reads were processed and aligned to the reference…
Plant Physiol. 2011 Jul;156(3):1257-68. doi: 10.1104/pp.110.169748. Epub 2011 Apr 29.PMID: 21531898
A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat.
Cristobal Uauy 1, Francine Paraiso, Pasqualina Colasuonno, Robert K Tran, Helen Tsai, Steve Berardi, Luca Comai, Jorge Dubcovsky
Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use…
BMC Plant Biol. 2009 Aug 28;9:115. doi: 10.1186/1471-2229-9-115.PMID: 19712486
TILLING to detect induced mutations in soybean.
Jennifer L Cooper 1, Bradley J Till, Robert G Laport, Margaret C Darlow, Justin M Kleffner, Aziz Jamai, Tarik El-Mellouki, Shiming Liu, Rae Ritchie, Niels Nielsen, Kristin D Bilyeu, Khalid Meksem, Luca Comai, Steven Henikoff
Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world’s protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).
BMC Plant Biol. 2008 Jan 24;8:9. doi: 10.1186/1471-2229-8-9.PMID: 18218134
Discovery of chemically induced mutations in rice by TILLING.
Bradley J Till 1, Jennifer Cooper, Thomas H Tai, Peter Colowit, Elizabeth A Greene, Steven Henikoff, Luca Comai
Rice is both a food source for a majority of the world’s population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method…
BMC Plant Biol. 2007 Apr 11;7:19. doi: 10.1186/1471-2229-7-19.PMID: 17428339
TILLING: practical single-nucleotide mutation discovery.
In the post-genomic sequencing era, an expanding portfolio of genomic technologies has been applied to the study of gene function. Reverse genetics approaches that provide targeted inactivation of genes identified by sequence analysis include TILLING (for Targeting Local Lesions IN Genomes). TILLING searches the genomes of mutagenized organisms for mutations in a chosen gene, typically single base-pair substitutions. This review covers practical aspects of the technology, ranging from building the mutagenized population to mutation discovery, and discusses possible improvements to current protocols and the impact of new genomic methods for mutation discovery in relation to the future of the TILLING approach.
Plant J. 2006 Feb;45(4):684-94. doi: 10.1111/j.1365-313X.2006.02670.x.PMID: 16441355
High-throughput TILLING for Arabidopsis.
Bradley J Till 1, Trenton Colbert, Christine Codomo, Linda Enns, Jessica Johnson, Steven H Reynolds, Jorja G Henikoff, Elizabeth A Greene, Michael N Steine, Luca Comai, Steven Henikoff
Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.
Methods Mol Biol. 2006;323:127-35. doi: 10.1385/1-59745-003-0:127.PMID: 16739573
A Protocol for TILLING and Ecotilling in Plants and Animals
Bradley J Till 1, Troy Zerr, Luca Comai, Steven Henikoff
We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease…
Till BJ, Zerr T, Comai L, Henikoff S. A protocol for TILLING and Ecotilling in plants and animals. Nat Protoc. 2006;1(5):2465‐2477. doi:10.1038/nprot.2006.329
Two Callose Synthases, GSL1 and GSL5, Play an Essential and Redundant Role in Plant and Pollen Development and in Fertility
Linda C Enns 1, Masahiro M Kanaoka, Keiko U Torii, Luca Comai, Kiyotaka Okada, Robert E Cleland
Callose, a beta-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1-12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers…
Enns LC, Kanaoka MM, Torii KU, Comai L, Okada K, Cleland RE. Two callose synthases, GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility. Plant Mol Biol. 2005;58(3):333‐349. doi:10.1007/s11103-005-4526-7
Discovery of induced point mutations in maize genes by TILLING.
Bradley J Till 1, Steven H Reynolds, Clifford Weil, Nathan Springer, Chris Burtner, Kim Young, Elisabeth Bowers, Christine A Codomo, Linda C Enns, Anthony R Odden, Elizabeth A Greene, Luca Comai, Steven Henikoff
Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community…
BMC Plant Biol. 2004 Jul 28;4:12. doi: 10.1186/1471-2229-4-12.PMID: 15282033
TILLING. Traditional mutagenesis meets functional genomics.
Steven Henikoff 1, Bradley J Till, Luca Comai
Most of the genes of an organism are known from sequence, but most of the phenotypes are obscure. Thus, reverse genetics has become an important goal for many biologists. However, reverse-genetic methodologies are not similarly applicable to all organisms. In the general strategy for reverse genetics that we call TILLING (for Targeting Induced Local Lesions in Genomes), traditional chemical mutagenesis is followed by high-throughput screening for point mutations. TILLING promises to be generally applicable. Furthermore, because TILLING does not involve transgenic modifications, it is attractive not only for functional genomics but also for agricultural applications…
Plant Physiol. 2004 Jun;135(2):630-6. doi: 10.1104/pp.104.041061. Epub 2004 May 21.PMID: 15155876 Free PMC article. Review.
Mismatch Cleavage by Single-Strand Specific Nucleases
Bradley J Till 1, Chris Burtner, Luca Comai, Steven Henikoff
We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using…
Till BJ, Burtner C, Comai L, Henikoff S. Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 2004;32(8):2632‐2641. Published 2004 May 11. doi:10.1093/nar/gkh599
Efficient discovery of DNA polymorphisms in natural populations by Ecotilling.
Luca Comai 1, Kim Young, Bradley J Till, Steven H Reynolds, Elizabeth A Greene, Christine A Codomo, Linda C Enns, Jessica E Johnson, Chris Burtner, Anthony R Odden, Steven Henikoff
We have adapted the mutation detection technology used in Targeting Induced Local Lesions in Genomes (TILLING) to the discovery of polymorphisms in natural populations. The genomic DNA of a queried individual is mixed with a reference DNA and used to amplify a target 1-kbp region of DNA with asymmetrically labeled fluorescent primers. After heating and annealing, heteroduplexes are nicked at mismatched sites by the endonuclease CEL I and cut strands are visualized using Li-cor gel analyzers. Putative polymorphisms detected in one fluorescence channel can be verified by appearance of the opposite cut strand in the other channel. We demonstrated the efficiency of this technology, called Ecotilling…
Plant J. 2004 Mar;37(5):778-86. doi: 10.1111/j.0960-7412.2003.01999.x.PMID: 14871304
Spectrum of Chemically Induced Mutations From a Large-Scale Reverse-Genetic Screen in Arabidopsis
Elizabeth A Greene 1, Christine A Codomo, Nicholas E Taylor, Jorja G Henikoff, Bradley J Till, Steven H Reynolds, Linda C Enns, Chris Burtner, Jessica E Johnson, Anthony R Odden, Luca Comai, Steven Henikoff
Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of approximately 1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method…
Greene EA, Codomo CA, Taylor NE, et al. Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics. 2003;164(2):731‐740.
Large-scale discovery of induced point mutations with high-throughput TILLING.
Bradley J Till 1, Steven H Reynolds, Elizabeth A Greene, Christine A Codomo, Linda C Enns, Jessica E Johnson, Chris Burtner, Anthony R Odden, Kim Young, Nicholas E Taylor, Jorja G Henikoff, Luca Comai, Steven Henikoff
TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms…
Genome Res. 2003 Mar;13(3):524-30. doi: 10.1101/gr.977903.PMID: 12618384
High-throughput TILLING for functional genomics.
Bradley J Till 1, Trenton Colbert, Rachel Tompa, Linda C Enns, Christine A Codomo, Jessica E Johnson, Steven H Reynolds, Jorja G Henikoff, Elizabeth A Greene, Michael N Steine, Luca Comai, Steven Henikoff
Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.
Methods Mol Biol. 2003;236:205-20. doi: 10.1385/1-59259-413-1:205.PMID: 14501067
Targeting Induced Local Lesions IN Genomes (TILLING) for Plant Functional Genomics
C M McCallum 1, L Comai, E A Greene, S Henikoff
One of the most important breakthroughs in the history of genetics was the discovery that mutations can be induced (Muller, 1930; Stadler, 1932). The high frequency with which ionizing radiation and certain chemicals can cause genes to mutate made it possible to perform genetic studies that were not feasible when only spontaneous mutations were available. As a result, much of our understanding of genetics of higher organisms is based upon studies utilizing induced mutations for analyzing gene function. Alkylating agents, which yield predominantly point mutations, have been especially valuable, since the resulting altered and truncated protein products help to precisely map gene and protein function. Because of the high mutational density and the great utility of point mutations, traditional chemical mutagenesis methods have continued to be popular in phenotypic screens despite the development of other mutagenic tools such as transposon mobilization (Bingham et al., 1981)…
McCallum CM, Comai L, Greene EA, Henikoff S. Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol. 2000;123(2):439‐442. doi:10.1104/pp.123.2.439
Targeted Screening for Induced Mutations
C M McCallum 1, L Comai, E A Greene, S Henikoff
With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized.
McCallum CM, Comai L, Greene EA, Henikoff S. Targeted screening for induced mutations. Nat Biotechnol. 2000;18(4):455‐457. doi:10.1038/74542